Widely planted crop germplasm often represents the most elite lines containing a combination of the yield, agronomic, and pest and disease resistance traits most desired by growers. Because of its combination of elite traits, this germplasm may serve to generate commercially available seed, and may also be used as a source or future plant breeding efforts. In some cases, this germplasm may often comprise a transgenic trait in addition to the elite traits it exhibits. Thus, there exists a need in the field of plant breeding for methods to use the full complement of the existing germplasm base of a crop in plant breeding, regardless of whether a transgene is present in any of the elite varieties that might be used for breeding experiments. Methods to further apply zygosity testing to improve the efficiency of plant breeding efforts are also needed.
Zygosity and copy-number testing based on real-time quantitative TAQMAN™ PCR (qRT-PCR) has been used to analyze transgenic plant progeny (e.g. Bubner and Baldwin, Plant Cell Rep. 23:263-271(2004)). This technique can allow detection and quantization of the copy number of a given DNA sequence in a plant genome, and requires only a small amount of plant tissue. The technique is much less labor intensive per sample the previously employed methods, such as Southern blotting, for detecting and quantitating transgene copy numbers. Nevertheless, it is notoriously sensitive to the concentration and purity of the starting template DNA, among other variables. As such, its use has been primarily as a first screen for the copy number of a transgene before selective breading is begun in earnest, with subsequent confirmation of results by other means such as Southern blotting. It is known to be suited, for instance, for identifying high copy number events that may be discarded early in a oxygenic plant event analysis prior to selected events being passed on to breeders. The method has not been used with single copy elite transgenic breeding material.
If a transgenic plant line is to be used in a breeding program, efficient methods for removing a transgene, or screening for its loss are desirable. Specific removal of transgene sequences, such as a gene encoding a selectable marker, has been reported, including the Cre/lox recombinase system (Hare and Chua Nature Biotechnol. 20:575-580(2002)). This method can specifically lead to removal of inserted transgenic DNA sequences from a transgenic plant, but lox site sequences necessarily still remain, flanking the site of the excised sequence. Screening for loss of a transgene by genetic segregation in progeny is another method that is widely known but requires substantial time and effort to achieve. Thus, more efficient methods to screen for loss of specific sequences are desirable, especially methods that ensure the complete removal of all inserted transgenic sequences.
A significant portion of recent soybean breeding has utilized lines containing the ROUNDUP READY® trait found in event 40-3-2, because possibly as much as 80-95% of the soybean germplasm offered for sale in the United States currently contains this transgenic event. This germplasm exhibits a set of agronomic traits superior to the non-transgenic A5403 parent line, and to many conventional (i.e. non-transgenic) soybean breeding lines. Thus these transgenic ROUNDUP READY® soybean varieties are themselves useful in plant breeding programs. In order to fully realize their usefulness, it is desirable to be able to identify progeny of these ROUNDUP READY® lines that retain the elite agronomic traits of the parental line except for glyphosate resistance derived from the transgene insert of event 40-3-2 and the associated transgenic sequences found in event 40-3-2.
The sequences of the functional transgene of soybean event 40-3-2 and its associated flanking plant DNA can be used to detect the presence of the transgene in progeny of 40-3-2. The DNA sequence flanking the functional transgenic insert in soybean event 40-3-2 has been characterized (Padgette et al. Crop Sci. 35:1451-1461 (1995); Windels et al. Eur. Food Res. Technol. 213:107-112(2001).
Regulatory approval in many countries for release and commercialization of a particular transgenic plant event may require disclosure of genomic DNA sequences flanking an inserted DNA, and a method to detect DNA specific or the event. Accordingly, some sequences flanking the functional insertion in soybean event 40-3-2 have been reported previously (Monsanto MSL-16646, available at http link for //archive.food.gov.uk/pdf_files/acnfp/dossier.pdf). A method of detection for corn comprising transgenic event NK603 has also been reported at http link //gmo-crl.jrc.it/detectionmethods.htm.